Immunogenic control of tumours and tumour cells

ABSTRACT

The present invention relates to the use of immunogenic peptides comprising a T-cell epitope derived from a tumour-associated antigen and a redox motif such as C—(X)2-[CST] or [CST]-(X)2-C in the treatment of a tumour or in the treatment or prevention of a tumour relapse, and in the manufacture of medicaments therefore.

FIELD OF THE INVENTION

The present invention relates to immunogenic peptides and their use in(immune) therapies for the eradication of tumours and tumour cells andprevention of tumour relapses.

BACKGROUND OF THE INVENTION

Many tumours express antigens which may serve as a target or therapy.Such antigens can be broadly divided into:

-   oncogenes, such as the MAGE identified in some melanomas;-   proto-oncogenes, such as cyclin D1 expressed on soft tissues    carcinomas such as those of the kidney or parathyroid, as well as in    multiple myeloma;-   virus-derived proteins, such as those from the Epstein-Barr virus in    some carcinomas and in some Hodgkin-type lymphomas;-   surviving factors, which are anti-apoptotic factors such as survivin    or bcl2; and-   clonotypic determinants, such as idiotypic determinants derived from    B cell receptor in follicular lymphomas or multiple myelomas or T    cell receptor determinants in T cell malignancies.

Specific recognition of such antigens, expressed exclusively orpredominantly in tumour cells, offers the potential of a selectiveelimination of such cells. Active immunisation with tumour-associatedantigens or derivatives, or adoptive transfer of cells expanded in vitrowith such tumour-associated antigens could in theory be of interest forthe therapy of tumours. Over recent years, many attempts to elicittumour-specific elimination by specific immunotherapy have beenpublished. These included active immunisation with, for example,idiotype-derived peptides, as well as adoptive transfer of T cellsexpanded in vitro by exposure to tumour cells. Although very promising,these therapeutic approaches had limited success and/or were associatedwith a high rate of relapse. Besides, the capacity of T cells to undergoexpansion in vitro remains limited, with much loss of effector cells byapoptosis induced by overstimulation. Essentially all the work carriedout in the field of immunotherapy of tumours during the last 15 yearshas been devoted to methods to elicit cytolytic CD8+ T cells able torecognise and lyse tumour cells in a MHC class I dependent presentationof a tumour-derived antigen. The possibility of designing efficientimmunotherapy through MHC class II presentation of tumour-derivedpeptides and CD4+ T cells has not been explored until very recently(Perez-Diez et al. (2007), Blood 109, 5346-5354). This is imputable toseveral factors, including the widespread belief that most tumours donot express MHC class II determinants and that the function of CD4+ Tcells does not predispose them to be potent anti-tumour cells. Theclassical view is that CD4+ T cells can help in providing help to Bcells to produce specific antibodies and that the production ofIFN-gamma by Th1 CD4+ T cells could reduce angiogenesis. More recently,the requirement of CD4+ T cells as a source of IL-2 to help CD8+ T cellsto acquire full maturation has been described.

Despite major advances in the field of cancer treatment, immunotherapyof tumours is still in its infancy. The potential selectivity of suchimmunotherapy, certainly when targeting tumour-specific antigens, is animportant advantage and may eliminate the sometimes severe side effectsobserved with e.g. chemotherapy. Therefore, any new strategy forimmunotherapeutic treatment of cancer would be welcomed.

SUMMARY OF THE INVENTION

The present invention relates to the treatment of a tumour and/or theprevention of a tumour relapse in a patient using at least one isolatedimmunogenic peptide comprising (i) a T-cell epitope derived from atumour-associated antigen and (ii) a [CST]-(X)2-[CST] motif, moreparticularly a C—(X)2-[CST] or [CST]-(X)2-C motif.

The present invention relates in one aspect to the use of at least oneisolated immunogenic peptide comprising (i) a T-cell epitope derivedfrom a tumour-associated antigen and (ii) a [CST]-(X)2-[CST] motif, moreparticularly a C—(X)2-[CST] or [CST]-(X)2-C motif for the manufacture ofa medicament for treating a tumour or for preventing or treating atumour relapse.

In a further aspect, the invention also covers the use of at least oneisolated immunogenic peptide comprising (i) a T-cell epitope derivedfrom a tumour-associated antigen and (ii) a [CST]-(X)2-[CST] motif, forthe manufacture of a medicament for inducing CD4+ regulatory T cellswhich are cytotoxic to cells presenting said tumour-associated antigen.

The invention generally relates to immunogenic peptides comprising aT-cell epitope derived from a tumour-associated antigen and (ii) aC—(X)2-[CST] or [CST]-(X)2-C motif for use in treating a tumour or forpreventing or treating a tumour relapse and/or inducing CD4+ regulatoryT cells which are cytotoxic to cells presenting said tumour-associatedantigen.

In any of the above uses said tumour-associated antigen may be chosenfrom oncogenes, proto-oncogenes, viral proteins, surviving factors orclonotypic determinants.

In any of the above uses, said C—(X)2-[CST] or [CST]-(X)2-C motif insaid immunogenic peptide may be adjacent to said T-cell epitope, or beseparated from said T-cell epitope by a linker. In particular, saidlinker consists of at most 7 amino acids.

In further embodiments of the immunogenic peptide in the above uses,said C—(X)2-[CST] or [CST]-(X)2-C motif does not naturally occur withina region of 11 amino acids N-terminally or C-terminally of the T-cellepitope in the tumour-associated antigen. In particular embodiments,said C—(X)2-[CST] or [CST]-(X)2-C motif is positioned N-terminally ofthe T-cell epitope. Further in particular, at least one X in said[CST]-(X)2-[CST] motif is Gly, Ala, Ser or Thr; Additionally oralternatively, at least one X in the C—(X)2-[CST] or [CST]-(X)2-C motifis His or Pro. In an additional specification at least one C in theC—(X)2-[CST] or [CST]-(X)2-C motif is methylated.

In yet further embodiments of the immunogenic peptide for use in theherein described applications, the immunogenic peptide further comprisesan endosomal targeting sequence. Any of the above immunogenic peptidesmay be produced by chemical synthesis or by recombinant expression.

A further aspect of the invention relates to methods for obtaining apopulation of tumour-associated antigen-specific regulatory T cells withcytotoxic properties, said methods comprising the steps of:

-   -   providing peripheral blood cells;    -   contacting said cells with an immunogenic peptide comprising (i)        a T-cell epitope derived from a tumour-associated antigen        and (ii) a [CST]-(X)2-[CST] motif, more particularly a        C—(X)2-[CST] or [CST]-(X)2-C motif; and    -   expanding said cells in the presence of IL-2.

A further method of the invention aims at obtaining a population oftumour-associated antigen-specific regulatory T cells with cytotoxicproperties, and such methods comprise the steps of:

-   -   providing an immunogenic peptide comprising (i) a T-cell epitope        derived from a tumour-associated antigen and (ii) a        [CST]-(X)2-[CST] motif, more particularly a C—(X)2-[CST] or        [CST]-(X)2-C motif;    -   administering said immunogenic peptide to a subject; and    -   obtaining said population of tumour-associated antigen-specific        regulatory T cells from said subject.

Populations of tumour-associated antigen-specific regulatory T cellswith cytotoxic properties obtainable by the above methods are also partof the invention, as well as their use for the manufacture of amedicament for preventing or treating a tumour or tumour relapse.

A further aspect of the invention relates to isolated immunogenicpeptides comprising a T-cell epitope from a tumour-associated antigenand, adjacent to said T-cell epitope or separated from said T-cellepitope by a linker, a [CST]-(X)2-[CST], more particularly aC—(X)2-[CST] or [CST]-(X)2-C motif.

Yet another aspect of the invention relates to the use of at least oneisolated immunogenic peptide for the manufacture of a medicament for(substantially) treating or preventing a B-cell tumour or relapse of aB-cell tumour, the immunogenic peptide comprising (i) a T-cell epitopederived from said B-cell tumour idiotype and (ii) a [CST]-(X)2-[CST]motif, more particularly C—(X)2-[CST] or [CST]-(X)2-C.

The invention also encompasses the use of at least one isolatedimmunogenic peptide for the manufacture of a medicament for treating aT-cell tumour or for treating or preventing relapse of a T-cell tumour,the immunogenic peptide comprising (i) a T-cell epitope derived from aT-cell CDR3 of said tumour and (ii) a [CST]-(X)2-[CST] motif, moreparticularly C—(X)2-[CST] or [CST]-(X)2-C.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “peptide” when used herein refers to a molecule comprising anamino acid sequence of between 2 and 200 amino acids, connected bypeptide bonds, but which can in a particular embodiment comprisenon-amino acid structures (like for example a linking organic compound).Peptides according to the invention can contain any of the conventional20 amino acids or modified versions thereof, or can containnon-naturally occurring amino acids incorporated by chemical peptidesynthesis or by chemical or enzymatic modification.

The term “epitope” when used herein refers to one or several portions(which may define a conformational epitope) of a protein or factor whichis/are specifically recognised and bound by an antibody or a portionthereof (Fab′, Fab2′, etc.) or a receptor presented at the cell surfaceof a B or T cell lymphocyte, and which is able, by said binding, toinduce an immune response.

The term “antigen” when used herein refers to a structure of amacromolecule comprising one or more hapten(s) and/or comprising T cellepitopes. Typically, said macromolecule is a protein or peptide (with orwithout polysaccharides) or made of proteic composition and comprisesone or more epitopes; said macromolecule can herein alternatively bereferred to as “antigenic protein” or “antigenic peptide”.

The term “tumour-associated antigen” refers to any protein, peptide orantigen associated with (carried by, produced by, secreted by, etc) atumour or tumour cell(s). Tumour-associated antigens may be (nearly)exclusively associated with a tumour or tumour cell(s) and not withhealthy normal cells or may be overexpressed (e.g., 10 times, 100 times,1000 times or more) in a tumour or tumour cell(s) compared to healthynormal cells. More particularly a tumour-associated antigen is anantigen capable of being presented (in processed form) by MHCdeterminants of the tumour cell. Hence, tumour-associated antigens arelikely to be associated only with tumours or tumour cells expressing MHCmolecules.

The term “T cell epitope” or “T-cell epitope” in the context of thepresent invention refers to a dominant, sub-dominant or minor T cellepitope, i.e., a part of an antigenic protein or factor that isspecifically recognized and bound by a receptor at the cell surface of aT lymphocyte. Whether an epitope is dominant, sub-dominant or minordepends on the immune reaction elicited against the epitope. Dominancedepends on the frequency at which such epitopes are recognised by Tcells and able to activate them, among all the possible T cell epitopesof a protein. In particular, a T cell epitope is an epitope bound by MHCclass I or MHC class II molecules.

The term “MHC” refers to “major histocompatibility antigen”. In humans,the MHC genes are known as HLA (“human leukocyte antigen”) genes.Although there is no consistently followed convention, some literatureuses HLA to refer to HLA protein molecules, and MHC to refer to thegenes encoding the HLA proteins. As such the terms “MHC” and “HLA” areequivalents when used herein. The HLA system in man has its equivalentin the mouse, i.e., the H2 system. The most intensely-studied HLA genesare the nine so-called classical MHC genes: HLA-A, HLA-B, HLA-C,HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, and HLA-DRB1. Inhumans, the MHC is divided into three regions: Class I, II, and III. TheA, B, and C genes belong to MHC class I, whereas the six D genes belongto class II. MHC class I molecules are made of a single polymorphicchain containing 3 domains (alpha 1, 2 and 3), which associates withbeta 2 microglobulin at cell surface. Class II molecules are made of 2polymorphic chains, each containing 2 chains (alpha 1 and 2, and beta 1and 2).

Class I MHC molecules are expressed on virtually all nucleated cells.Peptide fragments presented in the context of class I MHC molecules arerecognized by CD8+ T lymphocytes (cytotoxic T lymphocytes or CTLs). CD8+T lymphocytes frequently mature into cytotoxic effectors which can lysecells bearing the stimulating antigen. Class II MHC molecules areexpressed primarily on activated lymphocytes and antigen-presentingcells. CD4+ T lymphocytes (helper T lymphocytes or HTLs) are activatedwith recognition of a unique peptide fragment presented by a class IIMHC molecule, usually found on an antigen presenting cell like amacrophage or dendritic cell. CD4+ T lymphocytes proliferate and secretecytokines that either support an antibody-mediated response through theproduction of IL-4 and IL-10 or support a cell-mediated response throughthe production of IL-2 and IFN-gamma.

Functional HLAs are characterised by a deep binding groove to whichendogenous as well as foreign, potentially antigenic peptides bind. Thegroove is further characterised by a well-defined shape andphysico-chemical properties. HLA class I binding sites are closed, inthat the peptide termini are pinned down into the ends of the groove.They are also involved in a network of hydrogen bonds with conserved HLAresidues. In view of these restraints, the length of bound peptides islimited to 8-10 residues. However, it has been demonstrated thatpeptides of up to 12 amino acid residues are also capable of binding HLAclass I. Superposition of the structures of different HLA complexesconfirmed a general mode of binding wherein peptides adopt a relativelylinear, extended conformation.

In contrast to HLA class I binding sites, class II sites are open atboth ends. This allows peptides to extend from the actual region ofbinding, thereby “hanging out” at both ends. Class II HLAs can thereforebind peptide ligands of variable length, ranging from 9 to more than 25amino acid residues. Similar to HLA class I, the affinity of a class IIligand is determined by a “constant” and a “variable” component. Theconstant part again results from a network of hydrogen bonds formedbetween conserved residues in the HLA class II groove and the main-chainof a bound peptide. However, this hydrogen bond pattern is not confinedto the N-and C-terminal residues of the peptide but distributed over thewhole chain. The latter is important because it restricts theconformation of complexed peptides to a strictly linear mode of binding.This is common for all class II allotypes. The second componentdetermining the binding affinity of a peptide is variable due to certainpositions of polymorphism within class II binding sites. Differentallotypes form different complementary pockets within the groove,thereby accounting for subtype-dependent selection of peptides, orspecificity. Importantly, the constraints on the amino acid residuesheld within class II pockets are in general “softer” than for class I.There is much more cross reactivity of peptides among different HLAclass II allotypes. The sequence of the +/−9 amino acids of an MHC classII T cell epitope that fit in the groove of the MHC II molecule areusually numbered P1 to P9. Additional amino acids N-terminal of theepitope are numbered P-1, P-2 and so on, amino acids C-terminal of theepitope are numbered P+1, P+2 and so on.

The term “organic compound having a reducing activity” when used hereinrefers to compounds, more in particular amino acid sequences, capable ofreducing disulfide bonds in proteins. An alternatively used term is“redox motif”.

The term “therapeutically effective amount” refers to an amount of thepeptide of the invention or derivative thereof, which produces thedesired therapeutic or preventive effect in a patient. For example, inreference to a disease or disorder, it is the amount which reduces tosome extent one or more symptoms of the disease or disorder, and moreparticularly returns to normal, either partially or completely, thephysiological or biochemical parameters associated with or causative ofthe disease or disorder. According to one particular embodiment of thepresent invention, the therapeutically effective amount is the amount ofthe peptide of the invention or derivative thereof, which will lead toan improvement or restoration of the normal physiological situation. Forinstance, when used to therapeutically treat a mammal affected by animmune disorder, it is a daily amount peptide/kg body weight of the saidmammal. Alternatively, where the administration is through gene-therapy,the amount of naked DNA or viral vectors is adjusted to ensure the localproduction of the relevant dosage of the peptide of the invention,derivative or homologue thereof.

The term “natural” when used herein referring to a sequence relates tothe fact that the sequence is identical to a naturally occurringsequence or is identical to part of such naturally occurring sequence.In contrast therewith the term “artificial” refers to a sequence whichas such does not occur in nature. Unless otherwise specified the termsnatural and artificial thus exclusively relate to a particular aminoacid (or nucleotide) sequence (e.g. the sequence of the immunogenicpeptide, a sequence comprised within the immunogenic peptide, an epitopesequence) and do not refer to the nature of the immunogenic peptide assuch. Optionally, an artificial sequence is obtained from a naturalsequence by limited modifications such as changing one or more aminoacids within the naturally occurring sequence or by adding amino acidsN- or C-terminally of a naturally occurring sequence. Amino acids arereferred to herein with their full name, their three-letter abbreviationor their one letter abbreviation.

Motifs of amino acid sequences are written herein according to theformat of Prosite (Hulo et al. (2006) Nucleic Acids Res. 34 (Databaseissue D227-D230). The symbol X is used for a position where any aminoacid is accepted. Alternatives are indicated by listing the acceptableamino acids for a given position, between square brackets (‘[ ]’). Forexample: [CST] stands for an amino acid selected from Cys, Ser or Thr.Amino acids which are excluded as alternatives are indicated by listingthem between curly brackets (‘{ }’). For example: {AM} stands for anyamino acid except Ala and Met. The different elements in a motif areseparated from each other by a hyphen —. Repetition of an identicalelement within a motif can be indicated by placing behind that element anumerical value or a numerical range between parentheses. For example:X(2) corresponds to X—X, X(2, 4) corresponds to X—X or X—X—X or X—X—X—X, A(3) corresponds to A-A-A.

The term “homologue” when used herein with reference to the epitopesused in the context of the invention, refer to molecules having at least50%, at least 70%, at least 80%, at least 90%, at least 95% or at least98% amino acid sequence identity with the naturally occurring epitope,thereby maintaining the ability of the epitope to bind an antibody orcell surface receptor of a B and/or T cell. Particular embodiments ofhomologues of an epitope correspond to the natural epitope modified inat most three, more particularly in at most two, most particularly inone amino acid.

The term “derivative” when used herein with reference to the peptides ofthe invention refers to molecules which contain at least the peptideactive portion (i.e. capable of eliciting cytolytic CD4+ T cellactivity) and, in addition thereto comprises a complementary portionwhich can have different purposes such as stabilising the peptides oraltering the pharmacokinetic or pharmacodynamic properties of thepeptide.

The term “sequence identity” of two sequences when used herein relatesto the number of positions with identical nucleotides or amino acidsdivided by the number of nucleotides or amino acids in the shorter ofthe sequences, when the two sequences are aligned. In particularembodiments, said sequence identity is from 70% to 80%, from 81% to 85%,from 86% to 90%, from 91% to 95%, from 96% to 100%, or 100%.

The terms “peptide-encoding polynucleotide (or nucleic acid)” and“polynucleotide (or nucleic acid) encoding peptide” when used hereinrefer to a nucleotide sequence, which, when expressed in an appropriateenvironment, results in the generation of the relevant peptide sequenceor a derivative or homologue thereof. Such polynucleotides or nucleicacids include the normal sequences encoding the peptide, as well asderivatives and fragments of these nucleic acids capable of expressing apeptide with the required activity. According to one embodiment, thenucleic acid encoding the peptides according to the invention orfragment thereof is a sequence encoding the peptide or fragment thereoforiginating from a mammal or corresponding to a mammalian, mostparticularly a human peptide fragment.

The present invention provides strategies for immunotherapy of tumour ortumour cell(s) or tumour relapses using compounds comprising a T-cellepitope derived from a tumour-associated antigen to which a motif withthioreductase activity (or shortly: redox motif) is attached. Thesecompounds elicit tumour-associated antigen-specific CD4+ T-cells withstrong capacity to induce apoptosis of tumour cells. These cytotoxicCD4+ T-cells cells can be elicited in vivo by active immunisation withthese compounds or can be expanded in vitro (ex vivo) for adoptivetransfer into tumour-bearing hosts.

Thus, in one aspect the invention relates isolated immunogenic peptidesfor use in the treatment of a tumour or for the prevention of a tumourrelapse in a patient. More particularly the invention envisages the useof at least one isolated immunogenic peptide comprising (i) a T-cellepitope derived from a tumour-associated antigen and (ii) a C—(X)2-[CST]or [CST]-(X)2-C motif, for the manufacture of a medicament for treatinga tumour or for preventing or treating a tumour relapse.

In a further aspect, the invention also covers the use of at least oneisolated immunogenic peptide comprising (i) a T-cell epitope derivedfrom a tumour-associated antigen and (ii) a C—(X)2-[CST] or [CST]-(X)2-Cmotif, for the manufacture of a medicament for inducing CD4+ regulatoryT cells which are cytotoxic to cells presenting said tumour-associatedantigen.

In any of the uses described hereinabove, the subject or recipientreceiving said immunogenic peptide is a mammal, in particular a(non-human) primate or a human.

In any of the above uses a tumour-associated antigen may be chosen fromoncogenes, proto-oncogenes, viral proteins, surviving factors orclonotypic/idiotypic determinants. Such antigens are known and acceptedin the art. The first oncogenes associated with tumours were describedfor melanomas. The MAGE (melanoma-associated gene) products were shownto be spontaneously expressed by tumour cells in the context of MHCclass I determinants, and as such, recognised by CD8+ cytolytic T cells.However, MAGE-derived antigens, such as MAGE-3, are also expressed inMHC class II determinants and CD4+ specific T cells have been clonedfrom melanoma patients (Schutz et al. (2000) Cancer Research 60:6272-6275; Schuler-Thurner et al. (2002) J. Exp. Med. 195: 1279-1288).Peptides presented by MHC class II determinants are known in the art.Other examples include the gp100 antigen expressed by the P815mastocytoma and by melanoma cells (Lapointe (2001) J. Immunol. 167:4758-4764; Cochlovius et al. (1999) Int. J. Cancer, 83: 547-554).

Proto-oncogenes include a number of polypeptides and proteins which arepreferentially expressed in tumours cells, and only minimally in healthytissues. Cyclin D1 is cell cycle regulator which is involved in the G1to S transition. High expression of cyclin D1 has been demonstrated inrenal cell carcinoma, parathyroid carcinomas and multiple myeloma. Apeptide encompassing residues 198 to 212 has been shown to carry a Tcell epitope recognised in the context of MHC class II determinants(Dengiel et al. (2004) Eur. J. of Immunol. 34: 3644-3651).

Survivin is one example of a factor inhibiting apoptosis, therebyconferring an expansion advantage to survivin-expressing cells. Survivinis aberrantly expressed in human cancers of epithelial and hematopoieticorigins and not expressed in healthy adult tissues except the thymus,testis and placenta, and in growth-hormone stimulated hematopoieticprogenitors and endothelial cells. Interestingly, survivin-specific CD8+T cells are detectable in blood of melanoma patients. Survivin isexpressed by a broad variety of malignant cell lines, including renalcarcinoma, breast cancer, and multiple myeloma, but also in acutemyeloid leukemia, and in acute and chronic lymphoid leukemia (Schmidt(2003) Blood 102: 571-576). Other examples on inhibitors of apoptosisare Bcl2 and spi6.

Idiotypic determinants are presented by B cells in follicular lymphomas,multiple myeloma and some forms of leukemia, and by T cell lymphomas andsome T cell leukemias. Idiotypic determinants are part of theantigen-specific receptor of either the B cell receptor (BCR) or the Tcell receptor (TCR). Such determinants are essentially encoded byhypervariable regions of the receptor, corresponding tocomplementarity-determining regions (CDR) of either the VH or VL regionsin B cells, or the CDR3 of the beta chain in T cells. As receptors arecreated by the random rearrangement of genes, they are unique to eachindividual. Peptides derived from idiotypic determinants are presentedin MHC class II determinants (Baskar et al. (2004) J. Clin. Invest. 113:1498-1510). Some tumours are associated with expression of virus-derivedantigens. Thus, some forms of Hodgkin disease express antigens from theEpstein-Barr virus (EBV). Such antigens are expressed in both class Iand class II determinants. CD8+ cytolytic T cells specific for EBVantigens can eliminate Hodgkin lymphoma cells (Bollard et al. (2004) J.Exp. Med. 200: 1623-1633). Antigenic determinants such as LMP-1 andLMP-2 are presented by MHC class II determinants.

A minimum requirement for the cytotoxic CD4+ T-cells to be activated isto recognise a cognate tumour-associated antigen-derived epitopepresented by MHC class II determinants, leading to apoptosis of the APC.Expression of MHC class II determinants by tumour cells is likely to bemuch more frequent than previously thought. Thus, malignant cellsderived from the hematopoietic lineages and cells derived fromendothelium or epithelium progenitors express class II determinants. Inaddition, expression of such determinants can be induced by inflammatoryconditions that often prevail in tumours, as a result of the productionof cytokines such as IFN-gamma or TNF-alpha by host cells.

There may be situations in which more than one tumour-associated antigenexists in a given tumour or tumour cell. It is therefore anticipatedthat combination of two or more immunogenic peptides may be used for thetreatment of a tumour or for the treatment or prevention of a tumourrelapse.

In any of the uses and methods described hereinabove, the one or moreimmunogenic peptides can be replaced by CD4+ regulatory T-cells (Tregs)primed with the immunogenic peptide(s) (i.e., adoptive cell transfer),or can be replaced by a nucleotide sequence encoding the immunogenicpeptide(s) (e.g. in the form of naked DNA or a viral vector to beadministered to an individual instead of the immunogenic peptide). Inparticular, both active immunisation with immunogenic peptides andadoptive cell transfer of in vitro expanded Tregs can be envisaged forantigens which are associated with tumours and not with normal cells,namely oncogenes such as MAGE, idiotypic determinants and perhaps somevirus proteins. For tumour-associated antigens which are overexpressedin tumours but also present in healthy cells, adoptive cell transfer maybe the preferred option. It is further feasible to target tumour cellsby gene therapy, so as to express a given immunogenic peptide accordingto the invention only in tumour cells. In such a scenario, any tumourantigen can be used as starting point for designing an immunogenicpeptide according to the invention. In addition, a combination ofmultiple immunogenic peptides, i.e. more than 1 (e.g., 2, 3, 4, 5, 6, 7,8, 9, 10 or more), can be used in the above-described applications.These aspects of the invention, as well as the further modification ofthe immunogenic peptides are described in detail hereafter.

The present invention is based upon the finding that an immunogenicpeptide, comprising a T cell epitope derived from a tumour-associatedantigen and a peptide sequence, having reducing activity is capable ofgenerating a population of CD4+ regulatory T cells, which have acytotoxic effect on tumour-associated antigen presenting cells.

Accordingly, the invention relates to immunogenic peptides, whichcomprise at least one T-cell epitope of a tumour-associated antigen witha potential to trigger an immune reaction, coupled to an organiccompound having a reducing activity, such as a thioreductase sequencemotif. The T cell epitope and the organic compound are optionallyseparated by a linker sequence. In further optional embodiments theimmunogenic peptide additionally comprises an endosome targetingsequence (e.g. late endosomal targeting sequence) and/or additional“flanking” sequences.

The immunogenic peptides of the invention can be schematicallyrepresented as A-L-B or B-L-A, wherein A represents a T-cell epitope ofan antigen (self or non-self) with a potential to trigger an immunereaction, L represents a linker and B represents an organic compoundhaving a reducing activity.

The reducing activity of an organic compound can be assayed for itsability to reduce a sulfhydryl group such as in the insulin solubilityassay known in the art, wherein the solubility of insulin is alteredupon reduction, or with a fluorescence-labelled insulin. The reducingorganic compound may be coupled at the amino-terminus side of the T-cellepitope or at the carboxy-terminus of the T-cell epitope.

Generally the organic compound with reducing activity is a peptidesequence. Peptide fragments with reducing activity are encountered inthioreductases which are small disulfide reducing enzymes includingglutaredoxins, nucleoredoxins, thioredoxins and other thiol/disulfideoxidoreductases They exert reducing activity for disulfide bonds onproteins (such as enzymes) through redox active cysteines withinconserved active domain consensus sequences: C—X(2)-C, C—X(2)-S,C—X(2)-T, S—X(2)-C, T-X(2)-C (Fomenko et al. (2003) Biochemistry 42,11214-11225), in which X stands for any amino acid. Such domains arealso found in larger proteins such as protein disulfide isomerase (PDI)and phosphoinositide-specific phospholipase C.

Accordingly, in particular embodiments, immunogenic peptides accordingto the present invention comprise as redox motif the thioreductasesequence motif [CST]-X(2)-[CST], in a further embodiment thereto, said[CST]-X(2)-[CST] motif is positioned N-terminally of the T-cell epitope.More specifically, in said redox motif at least one of the [CST]positions is occupied by a Cys; thus the motif is either [C]—X(2)-[CST]or [CST]-X(2)-[C]. In the present application such a tetrapeptide willbe referred to as “the motif”. In particular embodiments peptides of theinvention contain the sequence motif [C]—X(2)-[CS] or [CS]—X(2)-[C]. Inmore particular embodiments peptides contain the sequence motifC—X(2)-S, S—X(2)-C or C—X(2)-C.

As explained in detail further on, the immunogenic peptides of thepresent invention can be made by chemical synthesis, which allows theincorporation of non-natural amino acids. Accordingly, in the motif ofreducing compounds according to particular embodiments of the presentinvention, C represents either cysteine or another amino acids with athiol group such as mercaptovaline, homocysteine or other natural ornon-natural amino acids with a thiol function. In order to have reducingactivity, the cysteines present in the motif should not occur as part ofa cystine disulfide bridge. Nevertheless, the motif may comprisemodified cysteines such as methylated cysteine, which is converted intocysteine with free thiol groups in vivo.

In particular embodiments of the invention, either of the amino acids Xin the [CST]-X(2)-[CST] motif of the immunogenic peptides of theinvention can be any natural amino acid, including S, C, or T or can bea non-natural amino acid. In particular embodiments X is an amino acidwith a small side chain such as Gly, Ala, Ser or Thr. In furtherparticular embodiments, X is not an amino acid with a bulky side chainsuch as Tyr. In further particular embodiments at least one X in the[CST]-X(2)-[CST] motif is His or Pro.

In the immunogenic peptides of the present invention comprising the(redox) motif described above, the motif is located such that, when theepitope fits into the MHC groove, the motif remains outside of the MHCbinding groove. The motif is placed either immediately adjacent to theepitope sequence within the peptide, or is separated from the T cellepitope by a linker. More particularly, the linker comprises an aminoacid sequence of 7 amino acids or less. Most particularly, the linkercomprises 1, 2, 3, or 4 amino acids. Alternatively, a linker maycomprise 6, 8 or 10 amino acids. Typical amino acids used in linkers areserine and threonine. Example of peptides with linkers in accordancewith the present invention are CXXC-G-epitope (SEQ ID NO:9),CXXC-GG-epitope (SEQ ID NO:10), CXXC—SSS-epitope (SEQ ID NO:11),CXXC—SGSG-epitope (SEQ ID NO:12) and the like. 2 0 In those particularembodiments of the peptides of the invention where the motif sequence isadjacent to the epitope sequence this is indicated as position P−4 toP−1 or P+1 to P+4 compared to the epitope sequence. Apart from a peptidelinker other organic compounds can be used as linker to link the partsof the immunogenic peptide to each other.

The immunogenic peptides of the present invention can further compriseadditional short amino acid sequences N or C-terminally of the(artificial) sequence comprising the T cell epitope and the reducingcompound (motif). Such an amino acid sequence is generally referred toherein as a ‘flanking sequence’. A flanking sequence can be positionedN- and/or C-terminally of the redox motif and/or of the T-cell epitopein the immunogenic peptide. When the immunogenic peptide comprises anendosomal targeting sequence, a flanking sequence can be present betweenthe epitope and an endosomal targeting sequence and/or between thereducing compound (e.g. motif) and an endosomal targeting sequence. Moreparticularly a flanking sequence is a sequence of up to 10 amino acids,or of in between 1 and 7 amino acids, such as a sequence of 2 aminoacids.

In particular embodiments of the invention, the redox motif in theimmunogenic peptide is located N-terminally from the epitope.

In further particular embodiments, where the redox motif present in theimmunogenic peptide contains one cysteine, this cysteine is present inthe motif in the position most remote from the epitope, thus the motifoccurs as C—X(2)-[ST] or C—X(2)-S N-terminally of the epitope or occursas [ST]-X(2)-C or S—X(2)-C carboxy-terminally of the epitope.

In certain embodiments of the present invention, immunogenic peptidesare provided comprising one epitope sequence and a motif sequence. Infurther particular embodiments, the motif occurs several times (1, 2, 3,4 or even more times) in the peptide, for example as repeats of themotif which can be spaced from each other by one or more amino acids(e.g. CXXC X CXXC X CXXC; SEQ ID NO:13), as repeats which are adjacentto each other (CXXC CXXC CXXC; SEQ ID NO:14) or as repeats which overlapwith each other CXXCXXCXXC (SEQ ID NO:15) or CXCCXCCXCC (SEQ ID NO:16)).Alternatively, one or more motifs are provided at both the N and the Cterminus of the T cell epitope sequence. Other variations envisaged forthe immunogenic peptides of the present invention include peptidescontaining repeats of a T cell epitope sequence or multiple differentT-cell epitopes wherein each epitope is preceded and/or followed by themotif (e.g. repeats of “motif-epitope” or repeats of“motif-epitope-motif”). Herein the motifs can all have the same sequencebut this is not obligatory. It is noted that repetitive sequences ofpeptides which comprise an epitope which in itself comprises the motifwill also result in a sequence comprising both the ‘epitope’ and a‘motif’. In such peptides, the motif within one epitope sequencefunctions as a motif outside a second epitope sequence. In particularembodiments however, the immunogenic peptides of the present inventioncomprise only one T cell epitope.

As described above the immunogenic peptides according to the inventioncomprise, in addition to a reducing compound/motif, a T cell epitopederived from a tumour-associated antigen. A T cell epitope in a proteinsequence can be identified by functional assays and/or one or more insilico prediction assays. The amino acids in a T cell epitope sequenceare numbered according to their position in the binding groove of theMHC proteins. In particular embodiments, the T-cell epitope presentwithin the peptides of the invention consists of between 8 and 25 aminoacids, yet more particularly of between 8 and 16 amino acids, yet mostparticularly consists of 8, 9, 10, 11, 12, 13, 14, 15 or 16 amino acids.In a more particular embodiment, the T cell epitope consists of asequence of 9 amino acids. In a further particular embodiment, theT-cell epitope is an epitope, which is presented to T cells by MHC-classII molecules. In particular embodiments of the present invention, the Tcell epitope sequence is an epitope sequence which fits into the cleftof an MHC II protein, more particularly a nonapeptide fitting into theMHC II cleft. The T cell epitope of the immunogenic peptides of theinvention can correspond either to a natural epitope sequence of aprotein or can be a modified version thereof, provided the modified Tcell epitope retains its ability to bind within the MHC cleft, similarto the natural T cell epitope sequence. The modified T cell epitope canhave the same binding affinity for the MHC protein as the naturalepitope, but can also have a lowered affinity. In particular embodimentsthe binding affinity of the modified peptide is no less than 10-foldless than the original peptide, more particularly no less than 5 timesless. It is a finding of the present invention that the peptides of thepresent invention have a stabilising effect on protein complexes.Accordingly, the stabilising effect of the peptide-MHC complexcompensates for the lowered affinity of the modified epitope for the MHCmolecule.

In particular embodiments, the immunogenic peptides of the inventionfurther comprise an amino acid sequence (or another organic compound)facilitating uptake of the peptide into (late) endosomes for processingand presentation within MHC class II determinants. The late endosometargeting is mediated by signals present in the cytoplasmic tail ofproteins and correspond to well-identified peptide motifs such as thedileucine-based [DE]XXXL[LI] (SEQ ID NO:17) or DXXLL (SEQ ID NO:18)motif (e.g. DXXXLL; SEQ ID NO:19), the tyrosine-based YXXØ motif or theso-called acidic cluster motif. The symbol Ø represents amino acidresidues with a bulky hydrophobic side chains such as Phe, Tyr and Trp.The late endosome targeting sequences allow for processing and efficientpresentation of the antigen-derived T cell epitope by MHC-class IImolecules. Such endosomal targeting sequences are contained, forexample, within the gp75 protein (Vijayasaradhi et al. (1995) J CellBiol 130, 807-820), the human CD3 gamma protein, the HLA-BM β (Copier etal. (1996) J. lmmunol. 157, 1017-1027), the cytoplasmic tail of theDEC205 receptor (Mahnke et al. (2000) J Cell Biol 151, 673-683). Otherexamples of peptides which function as sorting signals to the endosomeare disclosed in the review of Bonifacio and Traub (2003) Annu. Rev.Biochem. 72, 395-447. Alternatively, the sequence can be that of asubdominant or minor T cell epitope from a protein, which facilitatesuptake in late endosome without overcoming the T cell response towardsthe tumour-associated antigen -derived T cell epitope.

The immunogenic peptides of the invention can be generated by coupling areducing compound, more particularly a reducing motif as describedherein, N-terminally or C-terminally to a T-cell epitope of thetumour-associated antigen (either directly adjacent thereto or separatedby a linker). Moreover the T cell epitope sequence of the immunogenicpeptide and/or the redox motif can be modified and/or one or moreflanking sequences and/or a targeting sequence can be introduced (ormodified), compared to the naturally occurring T-cell epitope sequence.Accordingly, the resulting sequence of the immunogenic peptide will inmost cases differ from the sequence of the tumour-associated antigenprotein of interest. In this case, the immunogenic peptides of theinvention are peptides with an ‘artificial’, non-naturally occurringsequence.

The immunogenic peptides of the invention can vary substantially inlength, e.g. from about 12-13 amino acids (a T-cell epitope of 8-9 aminoacids and the 4-amino acid redox motif) to up to 50 or more amino acids.For example, an immunogenic peptide according to the invention maycomprise an endosomal targeting sequence of 40 amino acids, a flankingsequence of about 2 amino acids, a motif as described herein of 4 aminoacids, a linker of 4 amino acids and a T cell epitope peptide of 9 aminoacids. In particular embodiments, the immunogenic peptides of theinvention consist of between 12 amino acids and 20 up to 25, 30, 50, 75,100 or 200 amino acids. In a more particular embodiment, the peptidesconsist of between 10 and 20 amino acids. More particularly, where thereducing compound is a redox motif as described herein, the length ofthe immunogenic peptide comprising the epitope and motif optionallyconnected by a linker is 18 amino acids or less, e.g., 12, 13, 14, 15,16, 17, 18 or 19 amino acids.

As detailed above, the immunogenic peptides of the invention comprise areducing motif as described herein linked to a T cell epitope sequence.According to particular embodiments the T-cell epitopes are derived fromtumour-associated antigens which do not comprise within their nativenatural sequence an amino acid sequence with redox properties within asequence of 11 amino acids N- or C-terminally adjacent to the T-cellepitope of interest. Most particularly, the invention encompassesgenerating immunogenic peptides from tumour-associated antigens which donot comprise a sequence selected from C—X(2)-S, S—X(2)-C, C—X(2)-C,S—X(2)-S, C—X(2)-T, T-X(2)-C within a sequence of 11 amino acids N- orC-terminally adjacent to the epitope sequence. In further particularembodiments, the present invention provides immunogenic peptides oftumour-associated antigens which do not comprise the above-describedamino acid sequences with redox properties within their sequence.

In further particular embodiments, the immunogenic peptides of theinvention are peptides comprising T cell epitopes which T cell epitopesdo not comprise an amino acid sequence with redox properties withintheir natural sequence. However, in alternative embodiments, a T cellepitope binding to the MHC cleft may comprise a redox motif such asdescribed herein within its epitope sequence; the immunogenic peptidesaccording to the invention comprising such a T-cell epitope must furthercomprise another redox motif coupled (adjacent of separated by a linker)N- or C-terminally to the epitope such that the attached motif canensure the reducing activity (contrary to the motif present in theepitope, which is buried within the cleft).

Another aspect of the present invention relates to methods forgenerating immunogenic peptides of the present invention describedherein. Such methods include the identification of T-cell epitopes in atumour-associated antigen of interest; ways for in vitro and in silicoidentification T-cell epitopes are amply known in the art and someaspects are elaborated upon hereafter. In particular embodiments,methods according to the invention further include the generation ofimmunogenic peptides of the invention (including the identified T-cellepitope and a redox motif (with or without linker(s), flankingsequence(s) or endosomal targeting sequence)). The generated immunogenicpeptides may be assessed for the capability to induce tumour-associatedantigen-specific CD4+ regulatory T cells which are cytotoxic for cellspresenting (parts of) the tumour-associated antigen of interest.

Immunogenic peptides according to the invention are generated startingfrom T cell epitope(s) of the tumour-associated antigen(s) of interest.In particular, the T-cell epitope used may be a dominant T-cell epitope.The identification and selection of a T-cell epitope from atumour-associated antigen, for use in the context of the presentinvention is known to a person skilled in the art. For instance, peptidesequences isolated from a tumour-associated antigen are tested by, forexample, T cell biology techniques, to determine whether the peptidesequences elicit a T cell response. Those peptide sequences found toelicit a T cell response are defined as having T cell stimulatingactivity. Human T cell stimulating activity can further be tested byculturing T cells obtained from an individual sensitised to atumour-associated antigen with a peptide/epitope derived from thetumour-associated antigen and determining whether proliferation of Tcells occurs in response to the peptide/epitope as measured, e.g., bycellular uptake of tritiated thymidine. Stimulation indices forresponses by T cells to peptides/epitopes can be calculated as themaximum CPM in response to a peptide/epitope divided by the control CPM.A T cell stimulation index (S.I.) equal to or greater than two times thebackground level is considered “positive.” Positive results are used tocalculate the mean stimulation index for each peptide/epitope for thegroup of peptides/epitopes tested. Non-natural (or modified) T-cellepitopes can further optionally be tested for their binding affinity toMHC class II molecules. The binding of non-natural (or modified) T-cellepitopes to MHC class II molecules can be performed in different ways.For instance, soluble HLA class II molecules are obtained by lysis ofcells homozygous for a given class II molecule. The latter is purifiedby affinity chromatography. Soluble class II molecules are incubatedwith a biotin-labelled reference peptide produced according to itsstrong binding affinity for that class II molecule. Peptides to beassessed for class II binding are then incubated at differentconcentrations and their capacity to displace the reference peptide fromits class II binding is calculated by addition of neutravidin. Methodscan be found in for instance Texier et al., (2000) J. Immunology 164,3177-3184). The immunogenic peptides of the invention have a mean T cellstimulation index of greater than or equal to 2.0. An immunogenicpeptide having a T cell stimulation index of greater than or equal to2.0 is considered useful as a prophylactic or therapeutic agent. Moreparticularly, immunogenic peptides according to the invention have amean T cell stimulation index of at least 2.5, at least 3.5, at least4.0, or even at least 5.0. In addition, such peptides typically have apositivity index (P.I.) of at least about 100, at least 150, at leastabout 200 or at least about 250. The positivity index for a peptide isdetermined by multiplying the mean T cell stimulation index by thepercent of individuals, in a population of individuals sensitive to atumour-associated antigen (e. g., at least 9 individuals, at least 16individuals or at least 29 or 30, or even more), who have T cells thatrespond to the peptide (thus corresponding to the SI multiplied by thepromiscuous nature of the peptide/epitope). Thus, the positivity indexrepresents both the strength of a T cell response to a peptide (S.I.)and the frequency of a T cell response to a peptide in a population ofindividuals sensitive to a tumour-associated antigen. In order todetermine optimal T cell epitopes by, for example, fine mappingtechniques, a peptide having T cell stimulating activity and thuscomprising at least one T cell epitope as determined by T cell biologytechniques is modified by addition or deletion of amino acid residues ateither the N- or C-terminus of the peptide and tested to determine achange in T cell reactivity to the modified peptide. If two or morepeptides which share an area of overlap in the native protein sequenceare found to have human T cell stimulating activity, as determined by Tcell biology techniques, additional peptides can be produced comprisingall or a portion of such peptides and these additional peptides can betested by a similar procedure. Following this technique, peptides areselected and produced recombinantly or synthetically. T cell epitopes orpeptides are selected based on various factors, including the strengthof the T cell response to the peptide/epitope (e.g., stimulation index)and the frequency of the T cell response to the peptide in a populationof individuals.

Candidate antigens can be screened by one or more in vitro algorithms toidentify a T cell epitope sequence within an antigenic protein. Suitablealgorithms are described for example in Zhang et al. (2005) NucleicAcids Res 33, W180-W183 (PREDBALB); Salomon & Flower (2006) BMCBioinformatics 7, 501 (MHCBN); Schuler et al. (2007) Methods Mol Biol.409, 75-93 (SYFPEITHI); Dönnes & Kohlbacher (2006) Nucleic Acids Res.34, W194-W197 (SVMHC); Kolaskar & Tongaonkar (1990) FEBS Lett. 276,172-174 and Guan et al. (2003) Appl Bioinformatics 2, 63-66 (MHCPred).More particularly, such algorithms allow the prediction within anantigenic protein of one or more nonapeptide sequences which will fitinto the groove of an MHC II molecule.

The immunogenic peptides of the invention can be produced by recombinantexpression in, e.g., bacterial cells (e.g. Escherichia coli), yeastcells (e.g., Pichia species, Hansenula species, Saccharomyces orSchizosaccharomyces species), insect cells (e.g. from Spodopterafrugiperda or Trichoplusia ni), plant cells or mammalian cells (e.g.,CHO, COS cells). The construction of the therefore required suitableexpression vectors (including further information such as promoter andtermination sequences) involves standard recombinant DNA techniques.Recombinantly produced immunogenic peptides of the invention can bederived from a larger precursor protein, e.g., via enzymatic cleavage ofenzyme cleavage sites inserted adjacent to the N- and/or C-terminus ofthe immunogenic peptide, followed by suitable purification.

In view of the limited length of the immunogenic peptides of theinvention, they can be prepared by chemical peptide synthesis, whereinpeptides are prepared by coupling the different amino acids to eachother. Chemical synthesis is particularly suitable for the inclusion ofe.g. D-amino acids, amino acids with non-naturally occurring side chainsor natural amino acids with modified side chains such as methylatedcysteine. Chemical peptide synthesis methods are well described andpeptides can be ordered from companies such as Applied Biosystems andother companies. Peptide synthesis can be performed as either solidphase peptide synthesis (SPPS) or contrary to solution phase peptidesynthesis. The best-known SPPS methods are t-Boc and Fmoc solid phasechemistry which is amply known to the skilled person. In addition,peptides can be linked to each other to form longer peptides using aligation strategy (chemoselective coupling of two unprotected peptidefragments) as originally described by Kent (Schnolzer & Kent (1992) Int.J. Pept. Protein Res. 40, 180-193) and reviewed for example in Tam etal. (2001) Biopolymers 60, 194-205. This provides the tremendouspotential to achieve protein synthesis which is beyond the scope ofSPPS. Many proteins with the size of 100-300 residues have beensynthesised successfully by this method. Synthetic peptides havecontinued to play an ever-increasing crucial role in the research fieldsof biochemistry, pharmacology, neurobiology, enzymology and molecularbiology because of the enormous advances in the SPPS.

The physical and chemical properties of an immunogenic peptide ofinterest (e.g. solubility, stability) is examined to determine whetherthe peptide is/would be suitable for use in therapeutic compositions.Typically this is optimised by adjusting the sequence of the peptide.Optionally, the peptide can be modified after synthesis (chemicalmodifications e.g. adding/deleting functional groups) using techniquesknown in the art.

Accordingly, in yet a further aspect, the present invention providesmethods for generating tumour-associated antigen-specific cytotoxic Tcells (Tregs or CD4+ regulatory T-cells) either in vivo or in vitro (exvivo). In particular said T cells are cytotoxic towards any cellpresenting a tumour-associated antigen and are obtainable as a cellpopulation. The invention extends to such (populations of)tumour-associated antigen cytotoxic Tregs obtainable by the hereindescribed methods.

In particular embodiments, methods are provided which comprise theisolation of peripheral blood cells, the stimulation of the cellpopulation in vitro by contacting an immunogenic peptide according tothe invention with the isolated peripheral blood cells, and theexpansion of the stimulated cell population, more particularly in thepresence of IL-2. The methods according to the invention have theadvantage that higher numbers of Tregs are produced and that the Tregscan be generated which are specific for the tumour-associated antigen(by using a peptide comprising an antigen-specific epitope).Alternatively, tumour-associated antigen-specific cytotoxic T cells maybe obtained by incubation in the presence of APCs presenting atumour-associated antigen-specific immunogenic peptide according to theinvention after transduction or transfection of the APCs with a geneticconstruct capable of driving expression of such immunogenic peptide.Such APCs may in fact themselves be administered to a subject in need totrigger in vivo in said subject the induction of the beneficial subsetof cytotoxic CD4+ T-cells.

In an alternative embodiment, the Tregs can be generated in vivo, i.e.by the administration of an immunogenic peptide provided herein to asubject, and collection of the Tregs generated in vivo.

The tumour-associated antigen-specific regulatory T cells obtainable bythe above methods are of particular interest for use in the manufactureof a medicament for treating a tumour or for preventing or treating atumour relapse, i.e., for any of the above-described uses of theimmunogenic peptides of the invention, said peptides can be replaced bysaid tumour-associated antigen-specific Tregs. Both the use ofallogeneic and autogeneic cells is envisaged. Any method comprising theadministration of said tumour-associated antigen-specific Tregs to asubject in need (i.e., for treating a tumour or for preventing ortreating a tumour relapse) is also known as adoptive cell therapy. Tregsare crucial in immunoregulation and have great therapeutic potential.The efficacy of Treg-based immunotherapy depends on the Ag specificityof the regulatory T cells. Moreover, the use of Ag-specific Treg asopposed to polyclonal expanded Treg reduces the total number of Tregnecessary for therapy.

The present invention also relates to nucleic acid sequences encodingthe immunogenic peptides of the present invention and methods for theiruse, e.g., for recombinant expression or in gene therapy. In particular,said nucleic acid sequences are capable of expressing an immunogenicpeptides of the invention.

The immunogenic peptides of the invention may indeed be administered toa subject in need by using any suitable gene therapy method. In any useor method of the invention for the treatment of a tumour and/or fortreatment or prevention of a tumour relapse, immunisation with animmunogenic peptide of the invention may be combined with adoptive celltransfer of (a population of) Tregs specific for said immunogenicpeptide and/or with gene therapy. When combined, said immunisation,adoptive cell transfer and gene therapy can be used concurrently, orsequentially in any possible combination.

In gene therapy, recombinant nucleic acid molecules encoding theimmunogenic peptides can be used as naked DNA or in liposomes or otherlipid systems for delivery to target cells. Other methods for the directtransfer of plasmid DNA into cells are well known to those skilled inthe art for use in human gene therapy and involve targeting the DNA toreceptors on cells by complexing the plasmid DNA to proteins. In itssimplest form, gene transfer can be performed by simply injecting minuteamounts of DNA into the nucleus of a cell, through a process ofmicroinjection. Once recombinant genes are introduced into a cell, theycan be recognised by the cells normal mechanisms for transcription andtranslation, and a gene product will be expressed. Other methods havealso been attempted for introducing DNA into larger numbers of cells.These methods include: transfection, wherein DNA is precipitated withcalcium phosphate and taken into cells by pinocytosis; electroporation,wherein cells are exposed to large voltage pulses to introduce holesinto the membrane); lipofection/liposome fusion, wherein DNA is packedinto lipophilic vesicles which fuse with a target cell; and particlebombardment using DNA bound to small projectiles. Another method forintroducing DNA into cells is to couple the DNA to chemically modifiedproteins. Adenovirus proteins are capable of destabilizing endosomes andenhancing the uptake of DNA into cells. Mixing adenovirus to solutionscontaining DNA complexes, or the binding of DNA to polylysine covalentlyattached to adenovirus using protein crosslinking agents substantiallyimproves the uptake and expression of the recombinant gene.Adeno-associated virus vectors may also be used for gene delivery intovascular cells. As used herein, “gene transfer” means the process ofintroducing a foreign nucleic acid molecule into a cell, which iscommonly performed to enable the expression of a particular productencoded by the gene. The said product may include a protein,polypeptide, anti-sense DNA or RNA, or enzymatically active RNA. Genetransfer can be performed in cultured cells or by direct administrationinto mammals. In another embodiment, a vector comprising a nucleic acidmolecule sequence encoding an immunogenic peptide according to theinvention is provided. In particular embodiments, the vector isgenerated such that the nucleic acid molecule sequence is expressed onlyin a specific tissue. Methods of achieving tissue-specific geneexpression are well known in the art, e.g., by placing the sequenceencoding an immunogenic peptide of the invention under control of apromoter, which directs expression of the peptide specifically in one ormore tissue(s) or organ(s). Expression vectors derived from viruses suchas retroviruses, vaccinia virus, adenovirus, adeno-associated virus,herpes viruses, RNA viruses or bovine papilloma virus, may be used fordelivery of nucleotide sequences (e.g., cDNA) encoding peptides,homologues or derivatives thereof according to the invention into thetargeted tissues or cell population. Methods which are well known tothose skilled in the art can be used to construct recombinant viralvectors containing such coding sequences. Alternatively, engineeredcells containing a nucleic acid molecule coding for an immunogenicpeptide according to the invention may be used in gene therapy.

Where the administration of one or more peptides according to theinvention is ensured through gene transfer (i.e. the administration of anucleic acid which ensures expression of peptides according to theinvention in vivo upon administration), the appropriate dosage of thenucleic acid can be determined based on the amount of peptide expressedas a result of the introduced nucleic acid.

The medicament of the invention is usually, but not necessarily, a(pharmaceutical) formulation comprising as active ingredient at leastone of the immunogenic peptides of the invention, a (population of)Tregs specific for said immunogenic peptide or a gene therapeutic vectorcapable of expressing said immunogenic peptide. Apart from the activeingredient(s), such formulation will comprise at least one of a(pharmaceutically acceptable) diluent, carrier or adjuvant. Typically,pharmaceutically acceptable compounds (such as diluents, carriers andadjuvants) can be found in, e.g., a Pharmacopeia handbook (e.g. US-,European- or International Pharmacopeia). The medicament orpharmaceutical composition of the invention normally comprises a(prophylactically or therapeutically) effective amount of the activeingredient(s) wherein the effectiveness is relative to the condition ordisorder to be prevented or treated. In particular, the pharmaceuticalcompositions of the invention are vaccines for prophylactic ortherapeutic application.

The medicament or pharmaceutical composition of the invention may needto be administered to a subject in need as part of a prophylactic ortherapeutic regimen comprising multiple administrations of saidmedicament or composition. Said multiple administrations usual occursequentially and the time-interval between two administrations can varyand will be adjusted to the nature of the active ingredient and thenature of the condition to be prevented or treated. The amount of activeingredient given to a subject in need in a single administration canalso vary and will depend on factors such as the physical status of thesubject (e.g.,weight, age), the status of the condition to be preventedor treated, and the experience of the treating doctor, physician ornurse.

The term “diluents” refers for instance to physiological salinesolutions. The term “adjuvant” usually refers to a pharmacological orimmunological agent that modifies (preferably increases) the effect ofother agents (e.g., drugs, vaccines) while having few if any directeffects when given by themselves. As one example of an adjuvantaluminium hydroxide (alum) is given, to which an immunogenic peptide ofthe invention can be adsorbed. Further, many other adjuvants are knownin the art and can be used provided they facilitate peptide presentationin MHC-class II presentation and T cell activation. The term“pharmaceutically acceptable carrier” means any material or substancewith which the active ingredient is formulated in order to facilitateits application or dissemination to the locus to be treated, forinstance by dissolving, dispersing or diffusing the said composition,and/or to facilitate its storage, transport or handling withoutimpairing its effectiveness. They include any and all solvents,dispersion media, coatings, antibacterial and antifungal agents (forexample phenol, sorbic acid, chlorobutanol), isotonic agents (such assugars or sodium chloride) and the like. Additional ingredients may beincluded in order to control the duration of action of the activeingredient in the composition. The pharmaceutically acceptable carriermay be a solid or a liquid or a gas which has been compressed to form aliquid, i.e. the compositions of this invention can suitably be used asconcentrates, emulsions, solutions, granulates, dusts, sprays, aerosols,suspensions, ointments, creams, tablets, pellets or powders. Suitablepharmaceutical carriers for use in said pharmaceutical compositions andtheir formulation are well known to those skilled in the art, and thereis no particular restriction to their selection within the presentinvention. They may also include additives such as wetting agents,dispersing agents, stickers, adhesives, emulsifying agents, solvents,coatings, antibacterial and antifungal agents (for example phenol,sorbic acid, chlorobutanol), isotonic agents (such as sugars or sodiumchloride) and the like, provided the same are consistent withpharmaceutical practice, i.e. carriers and additives which do not createpermanent damage to mammals. The pharmaceutical compositions of thepresent invention may be prepared in any known manner, for instance byhomogeneously mixing, coating and/or grinding the active ingredients, ina one-step or multi-steps procedure, with the selected carrier materialand, where appropriate, the other additives such as surface-activeagents. They may also be prepared by micronisation, for instance in viewto obtain them in the form of microspheres usually having a diameter ofabout 1 to 10 μm, namely for the manufacture of microcapsules forcontrolled or sustained release of the active ingredients.

Immunogenic peptides, homologues or derivatives thereof according to theinvention (and their physiologically acceptable salts or pharmaceuticalcompositions all included in the term “active ingredients”) may beadministered by any route appropriate to the condition to be preventedor treated and appropriate for the compounds, here the immunogenicproteins to be administered. Possible routes include regional, systemic,oral (solid form or inhalation), rectal, nasal, topical (includingocular, buccal and sublingual), vaginal and parenteral (includingsubcutaneous, intramuscular, intravenous, intradermal, intraarterial,intrathecal and epidural). The preferred route of administration mayvary with for example the condition of the recipient or with thecondition to be prevented or treated.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tabletseach containing a predetermined amount of the active ingredient; as apowder or granules; as solution or a suspension in an aqueous liquid ora non-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion. The active ingredient may also bepresented as a bolus, electuary or paste. A tablet may be made bycompression or moulding, optionally with one or more accessoryingredients. Compressed tablets may be prepared by compressing in asuitable machine the active ingredient in a free-flowing form such as apowder or granules, optionally mixed with a binder, lubricant, inertdiluent, preservative, surface active or dispersing agent. Mouldedtablets may be made by moulding in a suitable machine a mixture of thepowdered compound moistened with an inert liquid diluent. The tabletsmay optionally be coated or scored and may be formulated so as toprovide slow or controlled release of the active ingredient therein.

A further aspect of the invention relates to isolated immunogenicpeptides comprising a T-cell epitope from a tumour-associated antigenand, adjacent to said T-cell epitope or separated from said T-cellepitope by a linker, a [CST]-(X)2-[CST] motif, more particularly aC—(X)2-[CST] or [CST]-(X)2-C motif. In particular embodiments, thetumour-associated antigen is selected from the group consisting ofoncogenes, viral antigens, surviving factors and clonotypic/idiotypicdeterminants. In further particular embodiments the epitope is anepitope whereby the tumour associated antigen does not naturallycomprise within a sequence of 11 amino acids N- or C-terminally adjacentto said epitope, a redox motif. Illustrative examples of tumourassociated antigens for which immunogenic peptides are envisaged arementioned below.

An idiotype is made of the ensemble of antigenic determinants carried bythe variable part of antibodies and, as described above, thesedeterminants are reiterated in BCR (corresponding to CDRs) and TCR(corresponding to CDR3). BCR of a B-cell and the antibodies secreted bythe same B-cell share idiotypic determinants. During uptake ofpolypeptides or proteins by B cells, parts of the BCR are processedtogether with the antigen and are presented by MHC class IIdeterminants. In tumour B-cells such as B cell lymphomas or myelomas,the B-cell receptor (or BCR) is most often directed towards an antigenof undetermined specificity (for example the MGUS syndrome: monoclonalgammopathy of unknown specificity). Hence a strategy to treat such typesof tumours or tumour cells comprises the induction (by immunisationand/or gene therapy) of CD4+ regulatory T-cells cytotoxic towards tumourBCR T-cell epitopes (or an idiotope thereof; together referred tohereinafter as tumour B-cell idiotype) or T-cell CDR3 T-cell epitope,and/or adoptive transfer of said cytotoxic CD4+ regulatory T-cells.Indeed, as T-cell epitopes modified by attaching a redox motif theretoinduce CD4+ T-cells to acquire the property of inducing apoptosis inAPCs presenting said T-cell epitope (natural or modified), tumour B-cellBCR T-cell epitopes (or an idiotope thereof) or tumour T-cell CDR3T-cell epitope modified by attaching a redox motif thereto are capableof inducing CD4+ T-cells that can drive said tumour B-cells or tumourT-cells into apoptosis.

Hence, a further aspect of the invention relates to the use of at leastone isolated immunogenic peptide comprising (i) a T-cell epitope derivedfrom a tumour B-cell idiotype and (ii) a [CST]-(X)2-[CST] motif, moreparticularly a C—(X)2-[CST] or [CST]-(X)2-C motif, for the manufactureof a medicament for (substantially) treating or preventing a B-celltumour or relapse of such B-cell tumour.

The invention further relates to the use of at least one isolatedimmunogenic peptide comprising (i) a T-cell epitope derived from atumour B-cell idiotype and (ii) a [CST]-(X)2-[CST] motif, moreparticularly a C—(X)2-[CST] or [CST]-(X)2-C motif, for the manufactureof a medicament for inducing in a recipient CD4+ regulatory T cellswhich are cytotoxic to said tumour B-cell.

Yet another aspect of the invention relates to the use of at least oneisolated immunogenic peptide comprising (i) a T-cell epitope derivedfrom a tumour T-cell CDR3 and (ii) a [CST]-(X)2-[CST] motif, moreparticularly a C—(X)2-[CST] or [CST]-(X)2-C motif, for the manufactureof a medicament for (substantially) treating or preventing a T-celltumour or relapse of such T-cell tumour.

The invention further relates to the use of at least one isolatedimmunogenic peptide comprising (i) a T-cell epitope derived from atumour T-cell CDR3 and (ii) a [CST]-(X)2-[CST] motif, for themanufacture of a medicament for inducing in a recipient CD4+ regulatoryT cells which are cytotoxic to said tumour T-cell.

Further particular embodiments of the invention relate to methods oftreating patients suffering from a tumour expressing a viral antigen,which methods comprise, administering to the patient, an immunogenicpeptide according to the invention comprising a T-cell epitope againstthe viral antigen and a redox motif as described herein. In particularembodiments the viral antigen is an antigen of a virus selected from thegroup consisting of herpes viruses, C-type viruses an B-type RNA mammarytumour viruses.

The present invention will now be illustrated by means of the followingexamples, which are provided without any limiting intention.Furthermore, all references described herein are explicitly includedherein by reference.

Examples Example 1 Cytotoxic Regulatory CD4+ T Cells are Elicited by inVivo Immunisation with a Peptide Comprising a Melanoma-AssociatedAntigen (MAGE-3) T Cell Epitope to Which a Thioreductase ConsensusSequence is Added

C57BI/6 mice (group 1) are immunised with 25 μg of a peptide containinga (natural) T cell epitope of MAGE-3 by 3 footpath injections in CFA/IFAmade at a fortnight interval. The sequence of the (natural) T-cellepitope corresponds to amino acids 258 to 266 of MAGE-3, namely:YRQVPGSDP (SEQ ID NO:1).

A second group of C57BI/6 mice (group 2) are immunised using the sameprotocol with the peptide of SEQ ID NO:1 to which a consensus motifexhibiting thioreductase activity (or shortly: redox motif) was added atthe amino-terminal end, namely: CHGCYRQVPGSDP (SEQ ID NO:2; redox motifunderlined; modified T-cell epitope)

Ten days after the last immunisation, the spleens of all mice arerecovered and CD4+ T cells are prepared by sorting on magnetic beads.

Spleen adherent cells prepared from naïve C57BI/6 mice are used asantigen-presenting cells (APC). Such APC (2×10⁷) are loaded with eitherpeptide of SEQ ID NO:1 or peptide of SEQ ID NO:2 (5 μg/mL) by an 1-hincubation followed by a wash.

CD4+ T cells obtained from either group 1 or group 2 mice are added tothe population of APCs and co-cultured for 24 h at 37° C. Cells are thenrecovered and incubated with a fluorescent-labelled anti-CD11c antibodyand with FITC-labelled annexin V as a marker of apoptosis. Finally,cells are analysed by Facs analysis.

These experiments demonstrate that a peptide of SEQ ID NO:2 can elicitCD4+ T cells with cytotoxic properties towards APCs presenting eitherthe natural MAGE-3 T-cell epitope (SEQ ID NO:1) or the modified MAGE-3T-cell epitope (SEQ ID NO:2).

Example 2 Cytotoxic Regulatory CD4+ T Cells are Elicited by in VivoImmunisation with a Peptide Comprising a Cyclin D1 T-Cell Epitope toWhich a Thioreductase Consensus Sequence is Added

C57BI/6 mice (group 1) are immunised with 25 μg of a peptide containinga (natural) T-cell epitope of cyclin D1 by 3 footpath injections inCFA/IFA made at a fortnight interval. The sequence of the peptidecorresponds to amino acids 185 to 193 of cyclin D1, namely: FVALCATDV(SEQ ID NO:3).

A second group of C57BI/6 mice (group 2) are immunized using the sameprotocol as above but with peptide of SEQ ID NO:3 to which a consensusmotif exhibiting thioreductase activity (or shortly: redox motif) isadded at the amino-terminal end, namely: CHGCFVALCATDV (SEQ ID NO:4;redox motif underlined; modified T-cell epitope).

Ten days after the last immunisation, the spleens of all mice arerecovered and CD4+ T cells are prepared by sorting on magnetic beads.

Spleen adherent cells prepared from naïve C57BI/6 mice are used asantigen-presenting cells (APC). Such APC (2×10⁷) are loaded with eitherpeptide of SEQ ID NO:3 or peptide of SEQ ID NO:4 (5 μg/mL) by an 1-hincubation followed by a wash.

CD4+ T cells obtained from either group 1 or group 2 mice are added tothe population of APCs and co-cultured for 24 h at 37° C. Cells are thenrecovered and incubated with a fluorescent-labelled anti-CD11c antibodyand with FITC-labelled annexin V as a marker of apoptosis. Finally,cells are analysed by Facs analysis.

These experiments demonstrate that a peptide of SEQ ID NO:4 can elicitCD4+ T cells with cytotoxic properties towards APCs presenting eitherthe natural cyclin D1 T-cell epitope (SEQ ID NO:3) or the modifiedcyclin D1 T-cell epitope (SEQ ID NO:4).

Example 3 Cytotoxic Regulatory CD4+ T Cells are Elicited by in VivoImmunisation with a Peptide Comprising a Survivin T Cell Epitope toWhich a Thioreductase Consensus Sequence is Added

C57BI/6 mice (group 1) are immunised with 25 μg of a peptide containinga (natural) T cell epitope of survivin by 3 footpath injections inCFA/IFA made at a fortnight interval. The sequence of the peptidecorresponds to amino acids 61 to 69 of survivin, namely: FKELEGWEP (SEQID NO:5).

A second group of C57BI/6 mice (group 2) are immunised using the sameprotocol with peptide of SEQ ID NO:5 to which a consensus motifexhibiting thioreductase activity (or shortly: redox motif) was added atthe amino-terminal end, namely: CHGCFKELEGWEP (SEQ ID NO:6; redox motifunderlined; modified T-cell epitope).

Ten days after the last immunisation, the spleens of all mice arerecovered and CD4+ T cells are prepared by sorting on magnetic beads.

Spleen adherent cells prepared from naïve C57BI/6 mice are used asantigen-presenting cells (APC). Such APC (2×10⁷) are loaded with eitherpeptide of SEQ ID NO:5 or peptide of SEQ ID NO:6 (5 μg/mL) by an 1-hincubation followed by a wash.

CD4+ T cells obtained from either group 1 or group 2 mice are added tothe population of APCs and co-cultured for 24 h at 37° C. Cells are thenrecovered and incubated with a fluorescent-labelled anti-CD11c antibodyand with FITC-labelled annexin V as a marker of apoptosis. Finally,cells are analysed by Facs analysis.

These experiments demonstrate that a peptide of SEQ ID NO:6 can elicitCD4+ T cells with cytotoxic properties towards APCs presenting eitherthe natural survivin T-cell epitope (SEQ ID NO:5) or the modifiedsurvivin T-cell epitope (SEQ ID NO:6).

Example 4 Cytotoxic Regulatory CD4+ T Cells are Elicited by in VivoImmunisation with a Peptide Comprising an Epstein-Barr LMP2 T CellEpitope to Which a Thioreductase Consensus Sequence is Added

BALB/c mice (group 1) are immunised with 25 μg of a peptide containing a(natural) T cell epitope of LMP2 from the Epstein-Barr virus by 3footpath injections in CFA/IFA made at a fortnight interval. Thesequence of the peptide corresponds to amino acids 167 to 175 of LMP2,namely: VASSYAAAQ (SEQ ID NO:7).

A second group of BALB/c mice (group 2) are immunised using the sameprotocol with peptide of SEQ ID NO:7 to which a consensus motifexhibiting thioreductase activity (or shortly: redox motif) is added atthe amino-terminal end, namely: CHGCVASSYAAAQ (SEQ ID NO:8; redox motifunderlined; modified T-cell epitope).

Ten days after the last immunisation, the spleens of all mice arerecovered and CD4+ T cells are prepared by sorting on magnetic beads.

Spleen adherent cells prepared from naïve BALB/c mice are used asantigen-presenting cells (APC). Such APC (2×10⁷) are loaded with eitherpeptide of SEQ ID NO:7 or peptide of SEQ ID NO:8 (5 μg/mL) by an 1-hincubation followed by a wash.

CD4+ T cells obtained from either group 1 or group 2 mice are added tothe population of APCs and co-cultured for 24 h at 37° C. Cells are thenrecovered and incubated with a fluorescent-labelled anti-CD11c antibodyand with FITC-labelled annexin V as a marker of apoptosis. Finally,cells are analysed by Facs analysis.

These experiments demonstrate that a peptide of SEQ ID NO:8 can elicitCD4+ T cells with cytotoxic properties towards APCs presenting eitherthe natural Epstein-Barr LMP2 T-cell epitope (SEQ ID NO:7) or themodified Epstein-Barr LMP2 T-cell epitope (SEQ ID NO:8).

1. A method of manufacturing a medicament comprising formulating amedicament comprising at least one isolated immunogenic peptide saidmedicament treating a tumour or for treating or preventing a tumourrelapse, the immunogenic peptide comprising (i) a T-cell epitope derivedfrom a tumour-associated antigen of said tumour and (ii) a C—(X)2-[CST]or [CST]-(X)2-C motif.
 2. The method of claim 1, said medicamentinducing CD4+ regulatory T cells which are cytotoxic to cells presentingsaid tumour-associated antigen.
 3. The method according to claim 1wherein said tumour-associated antigen is an oncogene, a proto-oncogene,a viral protein, a surviving factor or a clonotypic determinant.
 4. Themethod according to claim 1 wherein said C—(X)2-[CST] or [CST]-(X)2-Cmotif is adjacent to said T-cell epitope, or is separated from saidT-cell epitope by a linker.
 5. The method according to claim 4 whereinsaid linker consists of at most 7 amino acids.
 6. The method accordingto claim 1 wherein said C—(X)2-[CST] or [CST]-(X)2-C motif does notnaturally occur within a region of 11 amino acids N- or C-terminallyadjacent to the T-cell epitope in said tumour-associated antigen.
 7. Themethod according to claim 1 wherein said immunogenic peptide furthercomprises an endosomal targeting sequence.
 8. The method according toclaim 1 wherein said C—(X)2-[CST] or [CST]-(X)2-C motif is positionedN-terminally of the T-cell epitope.
 9. The method according to claim 1wherein at least one X in said C—(X)2-[CST] or [CST]-(X)2-C motif isGly, Ala, Ser or Thr.
 10. The method according to claim 1 wherein atleast one X in said C—(X)2-[CST] or [CST]-(X)2-C motif is His or Pro.11. The method according to claim 1 wherein at least one C in saidC—(X)2-[CST] or [CST]-(X)2-C motif is methylated.
 12. The methodaccording to claim 1 wherein said immunogenic peptide is produced bychemical synthesis or by recombinant expression.
 13. A method forobtaining a population of tumour-associated antigen-specific regulatoryT cells with cytotoxic properties, the method comprising the steps of:providing peripheral blood cells; contacting said cells in vitro with animmunogenic peptide comprising (i) a T-cell epitope derived from atumour-associated antigen and (ii) a C—(X)2-[CST] or [CST]-(X)2-C motif;and expanding said cells in the presence of Interleukin 2 (IL-2).
 14. Amethod for obtaining a population of tumour-associated antigen-specificregulatory T cells with cytotoxic properties, the method comprising thesteps of: providing an immunogenic peptide comprising (i) a T-cellepitope derived from a tumour-associated antigen and (ii) a C—(X)2-[CST]or [CST]-(X)2-C motif; administering said immunogenic peptide to asubject; and obtaining said population of tumour-associatedantigen-specific regulatory T cells from said subject.
 15. A populationof tumour-associated antigen-specific regulatory T cells with cytotoxicproperties obtainable by the method according to claim
 13. 16. A methodof manufacturing a medicament comprising formulating a medicamentcomprising the population of tumour-associated antigen-specificregulatory T cells according to claim 15 said medicament treating atumour or for treating or preventing a tumour relapse.
 17. An isolatedimmunogenic peptide comprising a T-cell epitope from a tumour-associatedantigen and, adjacent to said T-cell epitope or separated from saidT-cell epitope by a linker, a C—(X)2-[CST] or [CST]-(X)2-C motif.
 18. Amethod of manufacturing a medicament comprising formulating a medicamentcomprising at least one isolated immunogenic peptide comprising (i) aT-cell epitope derived from a tumour B-cell idiotype and (ii) aC—(X)2-[CST] or [CST]-(X)2-C motif, said medicament treating said B-celltumour or for treating or preventing relapse of said B-cell tumour. 19.A method of manufacturing a medicament comprising formulating amedicament comprising at least one isolated immunogenic peptidecomprising (i) a T-cell epitope derived from a tumour T-cell CDR3 and(ii) a [CST]-(X)2-[CST] motif, said medicament treating said T-celltumour or for treating or preventing relapse of said T-cell tumour.